The present invention relates generally to those apparatus and methods of analysis and investigation which utilize a solid support in the form of membranes or similar media upon which selected specimens are transferred to or otherwise placed for analysis and evaluations. More particularly, the present invention relates to and is suited for use in those areas of biotechnology and molecular biology that utilize membranes upon which selected specimens are deposited for analysis, investigation, hybridization, and the like, such specimens including molecules and molecule fragments of DNA, RNA, and proteins.
Many laboratory and analytical procedures involve the use of a sheet-like membranes, such as nitrocellulose, treated nitrocellulose, and similar materials, upon which one or more specimens are deposited with the membrane then subjected to further processing steps to analyze, identify, or isolate selected fragments of the specimens. For example, in the investigation of nucleic acids, the study of the structure and characteristics of DNA and RNA, and the function of selected enzymes in dividing DNA and RNA molecules into fragments of varying size, the use of sheet-like membranes, particularly those of nitrocellulose, are central to isolating selected fragments having certain characteristics. The membranes used are typically quite thin (e.g., 0.001 to 0.005 inch) and formed as rectangles or circular discs, an 82 mm. diameter being the most common for circular disc membranes. Various membrane-utilizing processes have been developed for the investigation of nucleic acids; these processes have in common the step of transferring or otherwise depositing DNA or RNA specimens onto a membrane. The membrane is then subjected to subsequent processing in accordance with the particular methodology of the process. For example, in one process, termed the "dot" blot procedure, fragments of DNA molecules of unlike size are separated, for example, by ultra-centrifuging or column chromatography, into separate samples of like size. The separate samples are then deposited onto a nitrocellulose membrane with each sample occupying a dot-like area on the membrane. In another process, termed the "Southern" blot procedure, fragments of DNA molecules of unlike size are electrophoretically separated into groupings of similar size. The fragments are then transferred to a nitrocellulose membrane for subsequent processing to produce a visible indication, for example, by autoradiograph, of the position on the membrane of the target fragments. Regardless of the particular transfer mechanism employed, the resulting membrane will have groupings of DNA molecule fragments bound thereto.
The membrane is then subjected to a number of fluid treatment steps to identify a particular grouping of target DNA fragments on the membrane. Typically, the transferred DNA fragments are thermally `fixed` to the membrane by heating at a selected temperature for a period of time sufficient to effect fixing. In order to locate a group of particular target fragments bound to the membrane, a solution of DNA or RNA `probe` fragments complementary to the target fragments is prepared with the probe fragments coupled to a radio-active tracer material. The membrane is then washed in the probe solution, for example, by immersion in a capped bottle or container or in a heat-sealed plastic bag containing the probe solution, for an incubation period sufficient to allow the radio-tagged probe fragments to hybridized with their complementary target fragments on the membrane. Once sufficient time for annealing has lapsed, the membrane is then washed and treated in a series of buffer solutions, such as ribonuclease, at differing temperatures and concentrations designed to removed the excess unhybridized probe solution. The resulting membrane is dried and retains only the original DNA fragments and the hybridized probe and target radio-tagged fragments. Thereafter, the membrane is processed to yield a visible indication of the location of the annealed target/probe molecules. Typically, the visible indication is obtained by laying the membrane against one side of a radiation sensitive film so that the film is exposed by beta particle radiation from the radioactive tag. The location of the hybridized probe/target molecule fragments on the membrane is revealed by the developed film.
Conventional membrane-dependent procedures are generally labor intensive and require a rather high level of skill to insure valid and reproducible results and minimize physical damage to or contamination of the membrane. Also, the use of membranes is not conducive to time and cost efficiencies that would allow transfer the membrane-based methodologies to clinical, industrial, and agricultural applications where cost and time effectiveness is imperative.
In applicant's cross-referenced patent application, apparatus and method are disclosed for the time and cost efficient treatment of membrane in which the membrane specimen is mounted between leaves of a fabric-like material and inserted into a sealable, flexible walled jacket to create flow passages or channels on opposite sides of the membrane with the probe and buffer solutions introduced and removed through appropriate ports. Numerous flow channels are created across the surfaces of the membrane by the fabric-like leaves to result in the cost and time efficient treatment of the membrane. While the apparatus and method disclosed in applicant's cross-referenced patent application is well suited for its intended purpose, the apparatus can be used to treat one or a small number of membranes at one time. While treatment of one or a small number of membranes meets the needs of many users, a class of users exists which has need to process a large number of membranes simultaneously, such users including research institutions where a large number of membranes must be screened in a time and cost efficient manner. For these users, an apparatus that is limited to treating one or a small number of membranes represents a less than optimum solution to volume screening situations.